We are interested in the cell biology of cells that make steroid hormones, and hope to follow labeled steroid substrates and products through these cells by autoradiography at the light and electron microscope levels, and thus show how these materials move within the cells during synthesis and secretion. By this means, we hope to gain information about the time course of these processes, the location of the enzymes (by using specific inhibitors), substrate pools, and perhaps sites of regulation. Since steroids and their precursors are diffusible substances, routine autoradiography is not feasible--the substances would be removed or at least displaced by the technique. Over the last few years the principal investigator has developed a method for cutting frozen thin sections of fresh-frozen tissue for electron microscopy. These sections are prepared under conditions that should maintain diffusible substances in place. We have now developed a method of carrying out autoradiography on this material that we hope will keep diffusible substances in place. The method involves coating the section with carbon to protect it from residual moisture in the emulsion film. There are also rather involved manipulations to protect the material from atmospheric moisture. We are currently testing the method in a model system, the uptake and transport of labeled testosterone by liver cells. If the method proves to be sound, we will apply it to steroid- secreting cells after a pulse administration of labeled substrate. Biochemical studies are underway to determine the most favorable steroid-secreting tissue and particular species, as well as the optimal conditions for administering the substrates.